Bottom Line:
Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACTThe Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

fig7: Reduced apoptosis and increased neuron numbers in the developing trigeminal ganglion of HIPK2−/− mutants. (A–D) At E11.5, the number of neurons undergoing apoptosis is similar in the trigeminal ganglion of wild-type (A) and HIPK2−/− mutants (B; arrowheads highlight apoptotic profiles that are neurofilament positive). In contrast, neuronal apoptosis in the trigeminal ganglion is significantly reduced in HIPK2−/− mutants at E13.5 (C and D). Bar, 20 μm. (E) Quantification of neuronal apoptosis in the trigeminal ganglion of wild-type and HIPK2−/− mutants from E10.5 to postnatal day 0 (P0). Although no difference is detected at E10.5 and E11.5, trigeminal ganglion in HIPK2−/− mutant show much reduced apoptosis at E13.5, E15.5 and P0 (P < 0.01, t test). (F) Number of neurons, as assessed by neurofilament-positive cells, is increased in HIPK2−/− mutants at E13.5 (0.01 < P < 0.025) and at P0 (P < 0.01, t test). Data represent mean ± SEM (n = 4).

Mentions:
Previous data have indicated that loss of Brn3a, TrkA, or Bcl-xL results in prominent cell death and dramatic loss of neurons in developing sensory ganglia (Motoyama et al., 1995; Huang et al., 1999a,b). Hence, the observed increase in the expression of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants should create a condition rendering HIPK2−/− sensory neurons more resistant to apoptosis during development (Ensor et al., 2001). To test this possibility, we determined the number of neurons undergoing apoptosis and the total number of neurons in the trigeminal ganglion of wild-type and HIPK2−/− mutants at different developmental stages. In the wild-type trigeminal ganglion, apoptotic profiles were detected as early as E10.5 and reached a maximum between E11.5 to E13.5, followed by a progressive decline at E15.5 and P0 (Fig. 7, A, C, and E; Huang et al., 1999b). Most of the apoptotic profiles stained positive for neurofilament, indicating that they are indeed neurons (Fig. 7 A, arrowheads). Whereas the number of apoptotic neurons was similar between wild type and HIPK2−/− at E10.5 and E11.5, there was significantly reduced apoptosis in the mutant ganglion at E13.5, E15.5, and P0 (Fig. 7, B, C, and E; 50–60% reduction, P < 0.01; t test). In agreement with this, there were significantly more neurons in the HIPK2−/− ganglion at E13.5 and P0 (0.01 < P < 0.025 at E13.5 and P < 0.01 at P0, n = 4, t test). Thus, these data indicate that loss of HIPK2 leads to up-regulation of Brn3a and its target genes and reduces apoptotic cell death during the development of sensory ganglia.

fig7: Reduced apoptosis and increased neuron numbers in the developing trigeminal ganglion of HIPK2−/− mutants. (A–D) At E11.5, the number of neurons undergoing apoptosis is similar in the trigeminal ganglion of wild-type (A) and HIPK2−/− mutants (B; arrowheads highlight apoptotic profiles that are neurofilament positive). In contrast, neuronal apoptosis in the trigeminal ganglion is significantly reduced in HIPK2−/− mutants at E13.5 (C and D). Bar, 20 μm. (E) Quantification of neuronal apoptosis in the trigeminal ganglion of wild-type and HIPK2−/− mutants from E10.5 to postnatal day 0 (P0). Although no difference is detected at E10.5 and E11.5, trigeminal ganglion in HIPK2−/− mutant show much reduced apoptosis at E13.5, E15.5 and P0 (P < 0.01, t test). (F) Number of neurons, as assessed by neurofilament-positive cells, is increased in HIPK2−/− mutants at E13.5 (0.01 < P < 0.025) and at P0 (P < 0.01, t test). Data represent mean ± SEM (n = 4).

Mentions:
Previous data have indicated that loss of Brn3a, TrkA, or Bcl-xL results in prominent cell death and dramatic loss of neurons in developing sensory ganglia (Motoyama et al., 1995; Huang et al., 1999a,b). Hence, the observed increase in the expression of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants should create a condition rendering HIPK2−/− sensory neurons more resistant to apoptosis during development (Ensor et al., 2001). To test this possibility, we determined the number of neurons undergoing apoptosis and the total number of neurons in the trigeminal ganglion of wild-type and HIPK2−/− mutants at different developmental stages. In the wild-type trigeminal ganglion, apoptotic profiles were detected as early as E10.5 and reached a maximum between E11.5 to E13.5, followed by a progressive decline at E15.5 and P0 (Fig. 7, A, C, and E; Huang et al., 1999b). Most of the apoptotic profiles stained positive for neurofilament, indicating that they are indeed neurons (Fig. 7 A, arrowheads). Whereas the number of apoptotic neurons was similar between wild type and HIPK2−/− at E10.5 and E11.5, there was significantly reduced apoptosis in the mutant ganglion at E13.5, E15.5, and P0 (Fig. 7, B, C, and E; 50–60% reduction, P < 0.01; t test). In agreement with this, there were significantly more neurons in the HIPK2−/− ganglion at E13.5 and P0 (0.01 < P < 0.025 at E13.5 and P < 0.01 at P0, n = 4, t test). Thus, these data indicate that loss of HIPK2 leads to up-regulation of Brn3a and its target genes and reduces apoptotic cell death during the development of sensory ganglia.

Bottom Line:
Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Affiliation:
Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACTThe Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.